Andy’Thomas’ Group’Presentaon’’ 103012€¦ · Andy’Thomas’ Group’Presentaon’’ 103012. ... High%Resolu,on.NMRTechniques.in.OrganicChemistry,’Timothy’D.W.’Claridge,’Elsevier’Science’Ltd

2D-‐NMR Techniques for the Research Organic Chemist

Andy Thomas

Group Presenta@on

10-‐30-‐12

Outline

•  Brief History of NMR (with some general theory)

•  Homonuclear Correla@ons: (1H-‐1H or 13C-‐13C etc…)

•  Heteronuclear Correla@ons: (1H-‐13C or 1H-‐19F etc…) •  Nuclear Overhauser: through space interac@ons

•  Exchange Correla@ons: equilibrium

1.   Through-‐Bond interac3ons

2. Through-‐Space interac3ons

3. Chemical Exchange

Key developments

3 Nobel prizes in physics, 2 Nobel prizes in chemistry and 1 Nobel prize in medicine (6 total)

•  1950s-‐Coupling constants used as structural tools

•  1960s-‐Signal averaging used to improve sensi@vity

•  1970s-‐Computer controlled instrumenta@on

•  1980s-‐2D NMR was developed

•  1990s-‐Pulse field gradients used

•  2000s-‐Flow NMR, high sensi@vity probes

High-‐Resolu,on NMR Techniques in Organic Chemistry, Timothy D.W. Claridge, Elsevier Science Ltd, UK, 1999; Chapter 1

Physics Prize 1952 for the development of new methods of nuclear magne@c precision (NMR)

Felix Boch Stanford 1905-‐1983

Edward M. Purcell Harvard 1912-‐1997

Richard R. Ernst Physics Prize 1991

for the development of new high resolu@on methods of nuclear magne@c precision (NMR)

2002 Chemistry Prize

determina@on of proteins

Prize in Medicine 2003 for the discovery of MRI

Paul C. Lauterbur

Peter Mansfield

The beginning of NMR (First Spectrum 1951)

Spectrum of Ethanol at 30 MHz

Arnold, J.T. Dharmab, S.S., Packard, M.E., J. Chem. Phys. 1951, 19, 507

High Resolu@on

Outline


•  Homonuclear Correla3ons: (1H-‐1H or 13C-‐13C etc…)

•  Heteronuclear Correla3ons: (1H-‐13C or 1H-‐19F etc…) •  Nuclear Overhauser: through space interac@ons

•  Exchange Correla@ons: equilibrium




Homonuclear Correla@ons (main techniques)

COSY: COrrela@on SpectroscopY TOCSY: TOtal Correla@on SpectroscopY 1D selec@ve TOCSY INADEQUATE: (C−C correla@ons)

1H-‐1H, 19F-‐19F etc.

13C-‐13C

•  COSY-‐90-‐Correla@ng coupled homonuclear spins. (2-‐3 bonds)

•  DQF-‐COSY-‐Correla@ng coupled homonuclear spins. Coupling Constant informa@on can be obtained. (2-‐3 bonds) •  TOCSY-‐ Correlates Spin systems


Introducing Two-‐Dimensional Methods

•  Exp. consist of RF pulses with delay periods. •  The dimensions refer to two frequency dimensions. (normally)

Typical Pulse sequence

90° 90° t2

t1

p1 p2

p1 = prepara@on p2 = mixing period t1 = @me between pulse t2 = detec@on period


��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

Is COSY Necessary? (quinine)

Can we use homonuclear decoupling? Very difficult in this case:

Overlapping peaks Also @me consuming

��

��

��

��

��

��

��

��

��

•  Complex mul@plets •  Similar J values

N

PN M

I H

F

RKS

UO

Q

N

OMe

OHG

LT

ED

B

AC

A B C D

E F G H I

J

KLM NO

P

Q RS

TU

Spectrum from Ian Fleming

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

N

PN M

I H

F

RKS

UO

Q

N

OMe

OHG

LT

ED

B

AC

A

C

A

COSY of quinine Spectrum from Ian Fleming

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

N

PN M

I H

F

RKS

UO

Q

N

OMe

OHG

LT

ED

B

AC

K LM NO P

Q RS TU

The most useful 2D technique for structure determina@on

Spectrum from Ian Fleming

��

��

��

�

A

B

CHCl3

C D

E F G H I

MeA

P U

MeB MeC MeD,E

MeF

V X Y

MeG MeH

MeI MeJ

TMS

Rifampicin (data from Ian Fleming)

OH

N NNMeB

NH

O MeD

MeH

OH

MeGHO

MeIAcO

O

OOMeFO

MeJMeAOOHOH

MeC

DF

J

G

PW

X

VU

E BRC

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

� ��


OH

N NNMeB

NH

O MeD

MeH

OH

MeGHO

MeIAcO

O

OOMeFO

MeJMeAOOHOH

MeC

DF

J

G

PW

X

VU

E BRC

��

��

��

��

��

��

��

��

��

��

��

�

��


B E

u

OH

N NNMeB

NH

O MeD

MeH

OH

MeGHO

MeIAcO

O

OOMeFO

MeJMeAOOHOH

MeC

DF

J

G

PW

X

VU

E BRC

Large Large

Large

medium

small

small

J

J

F

D

D C

Homonuclear Correla@ons (main techniques)

COSY: COrrela@on SpectroscopY TOCSY: TOtal Correla@on SpectroscopY 1D selec@ve TOCSY INADEQUATE: (C−C correla@ons)

1H-‐1H, 31P-‐31P etc.

•  COSY-‐90-‐Correla@ng coupled homonuclear spins. (2-‐3 bonds)

•  DQF-‐COSY-‐Correla@ng coupled homonuclear spins. Coupling Constant informa@on can be obtained. (2-‐3 bonds) •  TOCSY-‐ Correlates Spin systems


��

�

�

�

�

�

�

�

N

PN M

I H

F

RKS

UO

Q

N

OMe

OHG

LT

ED

B

AC

1D TOCSY

A

B C

D E

F G H I

KLM NO

P Q RS

TU

When should we use TOCSY? Spectrum from Ian Fleming

��

�

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

�

��

OH

N NNMeB

NH

O MeD

MeH

OH

MeGHO

MeIAcO

O

OOMeFO

MeJMeAOOHOH

MeC

DF

J

G

PW

X

V U E BU C


��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

OH

N NNMeB

NH

O MeD

MeH

OH

MeGHO

MeIAcO

O

OOMeFO

MeJMeAOOHOH

MeC

DF

J

G

PW

X

V U E BU C

B

B C

D

E


Outline

•  Brief History of NMR (The 2nd dimension explained)


•  Heteronuclear Correla3ons: (1H-‐13C or 1H-‐19F etc…) •  Exchange Correla@ons: equilibrium

•  Nuclear Overhauser: through space interac@ons




HMQC: Heteronuclear Mul@ple-‐Quantum Correla@on HSQC: Heteronuclear Single-‐Quantum Correla@on HMBC: Heteronuclear Mul@ple-‐Bond Correla@on HETCOR: HETeronuclear CORrela@on spectroscopy

Heteronuclear Correla@ons (main techniques)

•  HMQC-‐Correla@ng coupled heteronuclear spins. (used to iden@fy directly connected nuclei)

•  HSQC-‐Same as HMQC except resolu@on is bemer (takes longer)

•  HMBC-‐Correlates coupled spins across mul@ple bonds

•  HETCOR-‐Same as HMQC except is detected through low field channel.


HETCOR vs. HMQC

180° 90° t2

13C:

Δt1

90° 90°

1/2J 1/3J Decoupler on

δ1 δ2 1H:

HETCOR: (13C−1H COSY) •  collected in 13C Channel

•  increase resolu@on in 13C

•  CH3Br the carbon is split

•  (been replaced by HMQC)

90° 90°

t2

13C: Δt1

90° 180°

1/2J 1/2J

Decoupler on

1H:

acquire

acquire

HMQC: (1H−13C COSY) •  collected in 1H Channel

•  increase resolu@on in 1H

•  CH3CH2Br (1H−1H coupling)

•  Usually experiment of choice (Time)

HETCOR vs. HMQC

��

�

��

��

��

��

��

��

��

��

��

��

��

��

��

ONN

Ph

Ph Me

Me

H

H

��

��

�

�

��

��

��

��

��

��

��

��

�

�

��

��

��

��

��

��

SPh

O

O

CH3

Spectra from Tim and Alex

HMQC vs. HSQC

��

��

�

�

��

��

��

��

��

��

��

��

�

�

��

��

��

��

��

��

��

��

HMQC: Resolu@on not as good (@me is less) HSQC: Phase sensi@ve CH, CH3 vs. CH2

��

��

�

�

��

��

��

��

��

��

��

��

�

�

��

��

��

��

��

��

SPh

O

O

CH3

Spectra from Alex

Hybrid Experiments (HSQC-‐TOCSY)? Some@mes spectra are to crowded to analyze By adding TOCSY sequence aqer (HSQC) we can combine the experiments.

90° 90°

t2

13C: Δt1

90° 180°

1/2J 1/2J

Decoupler on

acquire TOCSY


HSQC-‐TOCSY


Can we Study mechanism with these methods? Tradi@onal 2D experiments take on average 7-‐45 min and up to hrs!!! (needs to be ultrafast) How can we monitor reac@ons in the NMR by HSQC, HMBC or TOCSY?

Int. Involved in reac@on iden@fied by (UF) TOCSY and HSQC

Group Problem

H

Herra, A., Fernandez-‐Valle, Mar@nez-‐Alvarez, Molero, D., Pardo, Z., Saez, E., Gal, M., Angew. Chem. 2009, 48, 6274

Can we Study mechanism with these methods? Tradi@onal 2D experiments take on average 7-‐45 min and up to hrs!!! (needs to be ultrafast) How can we monitor reac@ons in the NMR by HSQC, HMBC or TOCSY?

Int. Involved in reac@on iden@fied by UF TOCSY

Group Problem

H


Proposed Mechanism


UF TOCSY


UF TOCSY


Data from study


Outline



•  Heteronuclear Correla@ons: (1H-‐13C or 1H-‐19F etc…) •  Nuclear Overhauser: through space interac3ons

•  Exchange Correla3ons: equilibrium




NOESY: Nuclear Overhauser Effect

Where does NOE come from? (originate from dipolar coupling)

No coupling observed in solu@on due to rapid molecular movement

•  NOESY-‐Detects through space interac@ons •  EXSY-‐Same as NOESY experiment except chemical exchange is

observed •  ROESY-‐Is used to dis@nguish exchange from NOE


Where does the NOE come from?

Boltzmann distribu@on of spin states


NOE: Nuclear Overhauser Effect When a proton is saturated or inverted, spa@ally-‐close protons may experience an intensity enhancement which is termed Nuclear Overhauser Effect.

Small molecules NOE can be observed from about 4 angstroms apart Large molecules NOE can be observed from about 5 angstroms apart

Molecular Weight and Maximum NOE Maximum NOE depends on the molecular correla@on @me which (inverse rate of molecular tumbling) depends on the molecular weight and solvent viscosity. 1D vs 2D experiments

Time ≅ (30 min) good ≅ 1.5 hr (good) Informa@on irradiate one proton all interac@ons observed Spectra Crowding <30 Hz apart is problem no problem

2D 1D

Parameters needed for 2D NOESY: 1.  Tune at desired temp. (Important!!) 2.  PW 90° 3.  Find T1 4.  Select mixing @me (MOST Important)

small molecules 0.5−1 sec medium molecules 0.1−0.5 sec large molecules 0.05−0.3 sec

Normally one is only interested in interac@ons. If one is interested in measuring bond distances mixing @me is varied from experiment

Suggested Mixing @me

Applica3ons

Rela@ve configura@on of compounds can measure bond distances

N

PN M

I H

F

RKS

UO

Q

N

OMe

OHG

LT

ED

B

AC

Bums, C., Jones, C., Harvey, J., Chem. Comm. 2011, 47, -‐1193

Applica3ons

Rela@ve configura@on of compounds can measure bond distances Can iden@fy conforma@ons in solu@on (also typically need calcula@on to help support)

Bums, C., Jones, C., Harvey, J., Chem. Comm. 2011, 47, -‐1193

EXSY: EXchange SpectroscopY and NOE and ROE 2D

•  Experiment is homonuclear •  pulse sequence is equivalent to NOESY •  Integra@on of cross peaks is related to the equilibrium constant •  When finding equilibrium constants mul@ple experiments are performed varying mixing @me •  Technique has gained in popularity over the past few years in rate studies

If it is the same how do we dis3nguish between chemical exchange and NOE? •  You know you have interconver@ng species (EXSY) •  USE ROESY experiment

2D ROESY also (called CAMEL SPIN) the spinlock causes the spins to always be posi@ve unlike in the NOE experiment. This is used to dis3nguish NOE from exchange.

CAMEL SPIN: Cross-‐relaxa@on Appropriate for Minimolecules Emulated by Lock SPINs

Problem! TOCSY cross peaks can be observed

NOE NOE

ROE

exchange

ROESY

NOESY or EXSY

Exchange or TOCSY

Exchange

EXSY, NOESY and ROESY

Chemical Exchange (examples)

H3C

O

NCH3

CH3H3C

O

NCH3

CH3A

AB

B

O

CuCH3

CH3

B

A

No free rotation

O

B

ACH3

CuCH3

O

CuCH3

CH3 B

A

No free rotation

Hindered rota3on about a bond

Equilibrium

Rate of exchange can be measured by spin popula3on inversion

What types of experiments can be done? 1D−SPI: Spin Popula@on Inversion 2D−Exsy Exchange Spectroscopy

Good for simple cases

SPI: Spin Popula@on Inversion

H3C

O

NCH3

CH3 A

B

H3C

O

NCH3

CH3H3C

O

NCH3

CH3A

AB

B

What does this experiment accomplish?

One can measure rate of exchange and can calculate ac@va@on barriers (ΔH and ΔS)

What informa3on does one Need?

Need to obtain Pw 90 and T1 values at the desired temperature

How is the experiment conducted?

Probe tune

Jarek, R.L., Flesher, R.J., Shin, S.K., J. Chem. Educa,on. 1997, vol. 74, 978


H3C

O

NCH3

CH3 A

B

H3C

O

NCH3

CH3H3C

O

NCH3

CH3A

AB

B



H3C

O

NCH3

CH3 A

B

H3C

O

NCH3

CH3H3C

O

NCH3

CH3A

AB

B

•  [A] and [A*] lower and upper spin state (same for B) •  T1A and T1B are spin labce reac@on @mes •  MA = [A]−[A*] is the net magne@za@on •  k is the mutual-‐site exchange site rate constant



H3C

O

NCH3

CH3H3C

O

NCH3

CH3A

AB

B


-600

-400

-200

0

200

400

600

800

0 5 10 15 20 25 30 35

Inte

rgra

tion

Time (s)


10 °C Integra@on values with @me

1.  Measure 90° Pulse at 10 °C 2.  Measure T1 for MeA and MeB 3.  Invert the spin of MeB 4.  Repeat at different Temp

T1= 4.28 @ 10 °C T1= 4.66 @ 10 °C

Use this to calculate k

MA (τ ) =M0,A 1−100+ X200

"

#$

%

&'(1− e−2kτ )e−τ T1

()*

+,-

Use this to calculate ac@va@on energy

lnA = ΔSR

"

#$

%

&'+ ln

ekBTh

"

#$

%

&'



T1= 4.28 @ 10 °C T1= 4.66 @ 10 °C

lnA = ΔSR

"

#$

%

&'+ ln

ekBTh

"

#$

%

&'

Use this to calculate ac@va@on energy

k = Ae −Ea RT

Key Points 1.  Simple technique 2.  Exchange is on NMR @me scale 3.  How can this be used to study mechanism? (equilibrium constants, interconver@ng species)

Plot of ln(k/t) vs 1000/T(K)


Donna Blackmond Chemistry

Bures, J., Armstrong, A., Blackmond, D., JACS, 2012, 134, 6741

NOESY(EXSY) from Blackmond Work


EXSY experiment (and different temp)


k1=2.0 M-1s-1, k-1=0.085s-1; k2=0.49 s-1, k-2=28 M-1s-1; k3=k-3=0

EXSY Impact on kine3cs

Bertz, Carlin, Deadwyler, Murphy, Ogle and Seagle J. Am. Chem. Soc. (2002), 124(46), 13650-‐1.


Proposed Structure


EXSY experiment


EXSY Data

The rapid-injection 1H NMR data points for the reaction of cyclohexenone with lithiumdimethylcuprate at –100 °C, where the curves for the predicted rates of appearance of π-complexes are calculated from the EXSY rate constants.

Reac@on of cyclohexenone with Me2CuLi.LiI at –100°C: RI vs. EXSY


Conclusions

•  COSY, TOCSY, HMQC, HSQC, HMBC (great structure determina@on methods) –  New Ultra fast methods can be used to gain insight into mechanism

•  EXSY, NOESY, HOESY, equilibrium constants, aggrega@on and bond distances can be measured

•  ROESY can dis@nguish between exchange and NOE

Documents

Andy’Thomas’ Group’Presentaon’’ 103012€¦ · Andy’Thomas’ Group’Presentaon’’ 103012. ... High%Resolu,on.NMRTechniques.in.OrganicChemistry,’Timothy’D.W.’Claridge,’Elsevier’Science’Ltd